Method for treating an auditory neuropathy spectrum disorder

ABSTRACT

The present invention provides a method for treating a method for treating an auditory neuropathy spectrum disorder in a subject comprising transferring a transgene via an adeno-associated virus (AAV) vector to the subject; wherein the transgene is selected from the group consisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, AND OTOF.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62/737,406, filed on Sep. 27,2018, all of which are hereby expressly incorporated by reference intothe present application.

FIELD OF THE INVENTION

The present invention relates to a method for treating an auditoryneuropathy spectrum disorder.

BACKGROUND OF THE INVENTION

Hearing loss is the most common pediatric sensory defect: more than1/1000 children are affected by severe to profound sensorineural hearingimpairment (SNHI) [1]. Pediatric SNHI is composed of a plethora ofdisease entities. Among them, auditory neuropathy spectrum disorder(ANSD) is of special interest because of its unparalleled clinicalmanifestations. ANSD is not uncommon, accounting for approximately 7% ofpermanent childhood hearing loss and a significant (but as yetundetermined) proportion of adult impairment [2]. Patients with ANSDhave various degrees of hearing loss with poor speech perception that isout of proportion to their hearing levels [3]. Audiologically, ANSD ischaracterized by the preservation of normal outer hair cell function asevidenced by the presence of otoacoustic emissions (OAEs) and/orcochlear microphonics (CM), whereas the transmission of the auditorysignal to the brainstem is impaired as evidenced by abnormalsound-evoked potentials of auditory brainstem response (ABR), poorspeech perception and the absence of acoustic reflexes [3-5]. Thepathophysiology of ANSD has been proposed to involve an abnormalperipheral auditory system localized to the inner hair cells, theauditory nerve, or the synapse between them [6]. Etiologically, ANSDmight be caused by environmental insults, including infection duringpregnancy, prematurity, perinatal hypoxemia and neonatalhyperbilirubinemia [7, 8], or it might be the consequence of certainsyndromes, such as Charcot-Marie-Tooth disease [9] or cri-du-chatsyndrome [10]. The tendency of familial aggregation observed in someseries suggests that genetic factors may also be involved in thepathogenesis [6-8]. It has been estimated that approximately 40% of ANSDcases may have a genetic basis [11].

It is desirable to develop a new method for treating an auditoryneuropathy spectrum disorder.

BRIEF SUMMARY OF THE INVENTION

It was unexpectedly discovered in the present invention that an auditoryneuropathy spectrum disorder can be efficiently treated through a genetherapy via a vector comprising an Adeno-associated virus (AAV), calledas an AAV vector hereinafter.

The present invention provides a method for treating a method fortreating an auditory neuropathy spectrum disorder in a subjectcomprising transferring a transgene via an adeno-associated virus (AAV)vector to the subject; wherein the transgene is selected from the groupconsisting of Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, andOTOF.

In another aspect, the present invention provides a construct fordelivering a transgene to a subject suffering from an auditoryneuropathy spectrum disorder, which comprises an adeno-associated virus(AAV) and the nucleic acids designated a transgene; wherein thetransgene is selected from the group consisting of Pjvk, PCDH15, GJB2,DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one color drawing.Copies of this patent or patent application publication with colordrawing will be provided by the USPTO upon request and payment of thenecessary fee.

The drawings presenting the preferred embodiments of the presentinvention are aimed at explaining the present invention. It should beunderstood that the present invention is not limited to the preferredembodiments shown. The data in the figures and examples are shown asmean±standard deviation (SD). Significant differences are shown asfollows: *p<0.05, ***p<0.001.

FIG. 1 provides the genes identified to be associated with good and poorCI outcomes.

FIG. 2 provides an image of the auditory thresholds of Pjvk^(WT/WT) andPjvk^(G292R/G292R) mice. FIG. 2(A) shows that ABR thresholds weremeasured in 3-week-old and 6-week-old mice. Pjvk^(G292R/G292R) mice(red) showed progressive severe hearing loss as compared to Pjvk^(WT/WT)mice (blue) at all frequencies (n=10 for each group; thresholdsexpressed in mean±SD). FIG. 2(B) shows that ABR traces (clicks-stimuli)at 100 dB SPL were superimposed (Pjvk^(WT/WT), black;Pjvk^(G292R/G292R), red). Note that wave I-V in 3-week-oldPjvk^(G292R/G292R) mice showed increased latencies and reduced peakamplitudes. FIG. 2(C) shows the patterns of parvalbuminimmunolocalization in hair cells and spiral ganglion neurons ofPjvk^(G292R/G292R) mice. Degeneration of organ of Corti and spiralganglion neurons are indicated by arrows and circles, respectively.(Bar=50 μm).

FIG. 3 shows the construct of an AAV vector containing the gene—Pjvk.

FIG. 4 shows the ABR thresholds at 8, 16, and 32 kHz inAnc80L65.Pjvk—treated versus untreated Pjvk^(G292R/G292R) mice.

FIG. 5 provides the results of the Anc80-Pjvk gene therapy, showing thatthe circling behavior was reduced in treated mice.

FIG. 6 provides the results of the Anc80-Pjvk gene therapy, showing thatthe rotarod performance was improves in treated mice.

FIG. 7 provides the results of the Anc80-Pjvk gene therapy, showing thatthe swimming performance was improves in treated mice.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art.

1. Non-Syndromic Auditory neuropathy spectrum disorder (ANSD)

Hearing loss is the most common pediatric sensory defect: more than1/1000 children are affected by severe to profound sensorineural hearingimpairment (SNHI) [1]. Pediatric SNHI is composed of a plethora ofdisease entities. Among them, auditory neuropathy spectrum disorder(ANSD) is of special interest because of its unparalleled clinicalmanifestations. ANSD is not uncommon, accounting for approximately 7% ofpermanent childhood hearing loss and a significant (but as yetundetermined) proportion of adult impairment [2]. Patients with ANSDhave various degrees of hearing loss with poor speech perception that isout of proportion to their hearing levels [3]. Audiologically, ANSD ischaracterized by the preservation of normal outer hair cell function asevidenced by the presence of otoacoustic emissions (OAEs) and/orcochlear microphonics (CM), whereas the transmission of the auditorysignal to the brainstem is impaired as evidenced by abnormalsound-evoked potentials of auditory brainstem response (ABR), poorspeech perception and the absence of acoustic reflexes [3-5]. Thepathophysiology of ANSD has been proposed to involve an abnormalperipheral auditory system localized to the inner hair cells, theauditory nerve, or the synapse between them [6].

Etiologically, ANSD might be caused by environmental insults, includinginfection during pregnancy, prematurity, perinatal hypoxemia andneonatal hyperbilirubinemia [7, 8], or it might be the consequence ofcertain syndromes, such as Charcot-Marie-Tooth disease [9] orcri-du-chat syndrome [10]. The tendency of familial aggregation observedin some series suggests that genetic factors may also be involved in thepathogenesis [6-8]. It has been estimated that approximately 40% of ANSDcases may have a genetic basis [11].

The hearing loss levels in patients with ANSD vary from mild to profoundhearing loss, and their speech perception may be out of proportion tothe audibility changes. In addition, patients with auditory neuropathydo not typically derive much benefit from hearing aids. Some of thepatients are able to acquire speech and hearing without a hearing aidover time; some of them present well with hearing aids or cochlearimplants (CIs); and still a part of them did not develop well in speechor hearing despite under CI use. These features lead to difficulties inthe diagnosis and treatment for patients with ANSD in clinical practice.

2. Pjvk Mutation and Non-Syndromic ANSD

Mutation in Pjvk is a common cause of non-syndromic ANSD in humans.Delmaghani et al. identified mutations in the Pjvk gene in four familialcases of ANSD [12]. Two missense mutations were identified in thefamilies. The Pjvk gene produces a protein the researchers named“pejvakin”, which is expressed in the organ of Corti, the spiralganglion and the neuronal cell bodies of the cochlear nuclei, superiorolivary complex and the inferior colliculus of the afferent auditorypathway. The researchers believe pejvakin is crucial for auditory nervesignalling. Mutation in this gene appears to result in auditoryneuropathy due to a disruption in neuronal signalling along the auditorypathway. Cochlear function is intact in these patients.

The PCDH15 gene is a member of the cadherin superfamily. Family membersencode integral membrane proteins that mediate calcium-dependentcell-cell adhesion. it plays an essential role in maintenance of normalretinal and cochlear function. Mutations in this gene result in hearingloss and Usher Syndrome Type IF (USH1F). Extensive alternative splicingresulting in multiple isoforms has been observed in the mouse ortholog.

3. Gene Mutation and Poor CI Outcome

We identified patients with mutations in some genes, including Pjvk,PCDH15 GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF, usually exhibitexcellent long-term CI outcomes, probably because the effects of thesemutations were confined to the inner ear and the function of theauditory nerve is spared. Based on the results of the clinical trial inmulti-center studies for the purpose to identify the geneticdeterminants of poor CI outcomes in Taiwan, which enrolled more than 300children with CIs, we identified genetic variants which are associatedwith poor CI outcomes in 7 (58%) of the 12 cases; 4 cases had bi-allelicPCDH15 pathogenic mutations and 3 cases were homozygous for the Pjvkp.G292R variant. Mutations in the WFS1, GJB3, ESRRB, LRTOMT, MYO3A, andPOU3F4 genes were detected in 7 (23%) of the 30 matched controls. Theallele frequencies of PCDH15 and Pjvkvariants were significantly higherin the cases than in the matched controls (both P<0.001). In the 7 CIrecipients with PCDH15 or Pjvkvariants, otoacoustic emissions wereabsent in both ears, and imaging findings were normal in all 7 implantedears. PCDH15 or Pjvkvariant is associated with poor CI performance, yetchildren with PCDH15 or Pjvkvariants might show clinical featuresindistinguishable from those of other typical pediatric CI recipients.

4. Adeno-Associated Virus (AAV)

The use of viral vectors for inner ear gene therapy is receivingincreased attention for treatment of genetic hearing disorders. Mostanimal studies to date have injected viral suspensions into neonatalears, via the round window membrane. Achieving transduction of haircells, or sensory neurons, throughout the cochlea has proven difficult,and no studies showed an efficient transduction of sensory cells inadult ears while maintaining normal cochlear functions [13].

Adeno-associated virus (AAV) vectors have emerged as a gene-deliveryplatform with demonstrated safety and efficacy in a handful of clinicaltrials for monogenic disorders. However, limitations of the currentgeneration vectors often prevent broader application of AAV genetherapy. Efforts to engineer AAV vectors have been hampered by a limitedunderstanding of the structure-function relationship of the complexmultimeric icosahedral architecture of the particle. To developadditional reagents pertinent to further our insight into AAVs, Luk H.Vandenberghe labortory inferred evolutionary intermediates of the viralcapsid using ancestral sequence reconstruction. In-silico-derivedsequences were synthesized de novo and characterized for biologicalproperties relevant to clinical applications [14]. This effort led tothe generation of nine functional putative ancestral AAVs and theidentification of Anc80, the predicted ancestor of the widely studiedAAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapyvector for targeting liver, muscle, and retina. Recently, noveladeno-associated virus (AAV) serotypes, such as Anc80, have beenconfirmed as a promising delivery system for restoring the function ofinner ear sensory cells [15]. However, the efficiency of these new AAVsin targeting other pathological changes of the auditory/vestibularpathways remains unclear.

In one preferred embodiment of the invention, the AAV vector is an AAVvector comprising an Anc80 capsid protein as provided in WO2017/100791A1, also called as an AAV-Anc80 vector. The AAV-Anc80 vector wasconfirmed to be able to efficiently deliver nucleic acids to the innerear, e.g., cochlea, particularly the inner and outer hair cells (IHCsand OHCs) in the cochlea, which is an attractive target for gene therapyapproaches to intervene in hearing loss and deafness of variousetiologies, most immediately monogenic forms of inherited deafness.

In one more preferred embodiment of the invention, the AAV vector is asynthetic inner ear hair cell targeting adeno-associated virus (AAV)vector, wherein the vector encodes a capsid having at least about 85%sequence identity to Anc80, and comprises a promoter selected from thegroup consisting of an Espin promoter, a PCDH15 promoter, a PTPRQpromoter and a TMHS (LHFPL5) promoter that directs expression ofharmonin-a, harmonin-b, or harmonin-c polypeptide, as provided inWO2018/145111 A1.

5. Gene Therapy

We have completed genetic studies in more than 250 patients withcochlear implantation. Mutations in most genes are related to good CIoutcomes, but mutations in PCDH15 and Pjvk are associated withunfavorable CI outcomes (see FIG. 1). Animal studies are currentlyunderway to elucidate the pathogenetic mechanisms and to explore noveltherapeutic approaches. Some genes have been identified to be associatedwith good and poor CI outcomes, as shown in FIG. 1.

The invention is further illustrated by the following example, whichshould not be construed as further limiting.

Example

1. Animal Model

Genome-editing techniques were used to produce Pjvk p.G292R genetransgenic mice, in which the hearing characterization has beencompleted in the PjVk^(G292R/G292R) mouse with the c.874 G>A mutation.Pjvk^(G2921/G292R) mice showed significantly higher hearing thresholdsthan PjVk^(G292R/WT) and Pjvk^(WT/WT) mice at clicks, as well as 8 k, 16k and 32 kHz pure tones on auditory brainstem response (FIG. 2A).Further analysis of the waveforms revealed prolonged latencies of allthe ABR waves (FIG. 2B), indicating the presence of retrocochlearlesions. In accordance with the audiological findings, pathologicalchanges in hair cells and spiral ganglion neurons were observed inPjVkG^(2921/G292R) mice at 1 Month (FIG. 2C). In addition to the hearingphenotypes, Pjvk^(G2921/G292R) mice also demonstrated equilibriumdeficits suggesting pathologies involving both the vestibular organ(circling) and the central nervous system (nodding).

2. Gene Therapy

The full length sequence of Pjvk gene was cloned into the AAV expressionvector. The construct can be co-transfected with the AAV-helpler plasmid(Anc80L65) into the HEK293 cells and produced the Anc80 virus particles.The construct of AAV-Pjvk was shown in FIG. 3.

A nanoliter microinjection system (Nanoliter2000; World PrecisionInstruments) was used to load Anc80L65.Pjvk into the glass micropipette(10 mm in diameter). A total of 0.7 ul Anc80L65.Pjvk was injected intoround window membrane of inner ear of Pjvk p.G292R mice at P0-P3. Shamsurgeries were performed as above with Anc80L65-GFP as a negativecontrol virus. At P45, the treated mice showed lower auditory brainstemresponse thresholds and improved vestibular function as compared to thecontrol group. The findings suggested that the Anc80-directed genetherapy was an efficient delivery system for simultaneously introducinggenes to both the sensory cells of the inner ear and the neurons of thecentral auditory/vestibular pathways.

The Anc80 virus particles were injected into the round window membraneby the microinjection.

3. Audiological Evaluations

Mice were anesthetized with sodium pentobarbital (35 mg/kg) deliveredintraperitoneally and maintained in a head-holder within an acousticallyand electrically insulated and grounded test room. We used an evokedpotential detection system (Smart EP 3.90, Intelligent Hearing Systems,Miami, Fla., USA) to measure the thresholds of the auditory brainstemresponse (ABR) in mice. Click sounds as well as 8, 16, and 32 kHz tonebursts at varying intensity (from 10 to 130 dB SPL), were given to evokeABRs of mice. The response signals were recorded with a subcutaneousneedle electrode inserted ventrolaterally into the ears of the mice.

The results of the gene therapy were shown in FIG. 4, showing that thecochlear function was rescued. The Pjvk^(G2921/G292R) mice treated withan AAV vector comprising Anc80 (Anc80-Pjvk) provided lower ABRthresholds measured at 6 weeks showed improved hearing thresholds in thetreated ears as compared with the untreated mice, especially at 8, 16and 32 kHz.

4. Vestibular Evaluations

Mice were subjected to a battery of vestibular evaluations, includingobservation of their circling behavior and head-tilting (performed atP90-120), a reaching test, a swimming test, and a rotarod test (allperformed at P90-120 age). The reaching responses of mice were recordedafter suspending animals by their tails and observing the reaction oftheir limbs and head-bobbing behaviors. Mice that extended theirforelimbs and tried to reach a surface were considered normal (i.e., interms of reaching response), whereas animals that either clasped theirforelimbs or exhibited head-bobbing behavior were classified asabnormal. For the swimming test, mice were observed for 15-20 s, andthose that maintained themselves well at the water surface wereclassified as normal, whereas those that failed to stay near the surfacewere considered abnormal. For the gripping test, mice were placed on thelower end of a 45-cm-long metal stick, and those that required more than15 s to reach the top of the stick were classified as abnormal (i.e.,<15 s were normal). For the rotarod test, mice were assessed for theirability to balance on a revolving rod (i.e., rotarod) of 3.5 cmdiameter. For each test, the mouse was placed on the rod rotating at 35rpm, and the time required for the mouse to fall was recorded. Eachmouse was tested 5 times, and the results were averaged.

5. Circling Behavior

The circling behavior of mice was quantified using optical tracking. A38-cm×58-cm box was attached to a video camera (gopro). The ImageJsoftware was set to track the head of mice placed within the box. Eachmouse was placed into the box and allowed to acclimate to the newenvironment for 2 min. Complete rotations were recorded and quantifiedfor the next 2 min, followed by a 1-min “cool-down” period in whichrotations were not tracked. Each mouse was assessed three times on thesame day, and the average was taken.

As shown in FIG. 5, the Pjvk mutant mice had significant vestibuledysfunction, as evidenced by their circling behavior. It was confirmedthat Anc80-Pjvk gene therapy reduced circling behavior in the micetreated with the gene therapy. After the gene therapy, the P90-120 micewas sent to the 30 sec-duration tracking of treated mice and untreated(mutant) control mice. The untreated mice had developed an obviouscircling behavior and showed poor ability to reach the field boundary(FIG. 5, left). On the contrary, the treated mice did not show thecircling behavior and could arrive the most boundary of the testingfield without the interruption of moving direction by circling (FIG. 5,right).

It was also confirmed in the rotarod test that the gene therapy improvedthe balance function in rotarod test. The rotarod test is a measure ofbalance function in which mice are placed on a rod that rotates withincreasing velocity, and the length of time the mouse remaining balancedon the rotating rod is recorded. The Pjvk mutant mice also showeddiminished balance on the rotarod test. After gene therapy, the treatedmice exhibited a better ability to stay on the rotating rod, and theuntreated control mice showed a poor ability to stay on the rod with ashorter retaining time (65.1±13.1 sec) than the treated mice (213.4±12.3 sec), see Table 1 and FIG. 6.

TABLE 1 The result of rotarod test at P90~120 Pjvk Pjvk Pjvk Normaluntreated treated w/ treated w/ control control Anc80-Pjvk Anc80-GFP (n= 6) (n = 8) (n = 8) (n = 5) Time on 213.2 ± 7.2 65.1 ± 13.1 213.4 ±12.3 79.4 ± 7.7 rod (sec)

6. Swim Testing

Mice were placed in a large container filled with room temperaturewater. Their swimming behavior was recorded using a video camera over 2min. The videos were de-identified and scored by an observer who wasblinded to the genotype and treatment and who was also not involved withthe initial video recording. A well-established 0-3 scoring system wasused to assess the swim performance. Briefly, a score of 0 indicatesnormal swimming behavior. A score of 1 indicates mild swimmingabnormality (circling, vertical swimming). A score of 2 indicatesmoderate swimming abnormality (immobile floating). A score of 3indicates significant swimming abnormality in which the mouse needs tobe rescued immediately (underwater tumbling). Swimming testing wasperformed at P90˜120 in all animals (6 normal control mice, 8 untreatedcontrol mice, 8 mutant mice that received Anc80-Pjvk gene therapy and 5mutant mice that received Anc80-GFP), see FIG. 7.

7. Inner Ear Morphology Studies

Tissues from inner ears of mice were subjected to hematoxylin and eosin(H&E) staining, and the morphology of each sample was examined with aLeica optical microscope. For light microscopy, inner ears from adultmice were fixed by perilymphatic perfusion with 4% paraformaldehyde(PFA) in phosphate-buffered saline (PBS) through round and oval windowsand a small fenestra in the apex of the cochlear bony capsule. Specimenswere subsequently rinsed in PBS buffer and decalcified in 4% PFA with0.35 M EDTA at 4° C. for 1 week. For light microscopy studies, sampleswere dehydrated and embedded in paraffin. Subsequently, serial sections(7 μm) were stained with H&E.

Whole-mount studies of mouse inner ear specimens were performed aspreviously described

with some minor modification. Briefly, after perfusion with 4% PFA, thecochleae were postfixed in the same solution for 2 h at room temperatureand washed in PBS. Segments of stria vascularis and organ of Cortitogether with Reissner's membrane were dissected out of the inner earspecimens using a fine needle. Samples were permeabilized in 1% TritonX-100 for 30 min and washed with PBS, followed by overnight incubationat 4° C. in blocking solution. The tissues were then stained withrhodamine-phalloidin (1:100 dilution; Molecular Probes, Eugene, Oreg.,USA). After washing in PBS, the tissues were mounted using the ProLongAntifade kit (Molecular Probes, Eugene, Oreg., USA) for 20 min at roomtemperature. Images of tissues were obtained using a laser scanningconfocal microscope (Zeiss LSM 510, Germany).

While the present invention has been disclosed by way preferredembodiments, it is not intended to limit the present invention. Anyperson of ordinary skill in the art may, without departing from thespirit and scope of the present invention, shall be allowed to performmodification and embellishment. Therefore, the scope of protection ofthe present invention shall be governed by which defined by the claimsattached subsequently.

REFERENCE

[1] J. B. Nadol, Jr., Hearing loss, N Engl J Med 329(15) (1993)1092-102.

[2] G. Rance, Auditory neuropathy/dys-synchrony and its perceptualconsequences, Trends Amplif 9(1) (2005) 1-43.

[3] A. Starr, T. W. Picton, Y. Sininger, L. J. Hood, C. I. Berlin,Auditory neuropathy, Brain 119 (Pt 3) (1996) 741-53.

[4] G. Rance, D. E. Beer, B. Cone-Wesson, R. K. Shepherd, R. C. Dowell,A. M. King, F. W. Rickards, G. M. Clark, Clinical findings for a groupof infants and young children with auditory neuropathy, Ear Hear 20(3)(1999) 238-52.

[5] M. Rodriguez-Ballesteros, R. Reynoso, M. Olarte, M. Villamar, C.Morera, R. Santarelli, E. Arslan, C. Meda, C. Curet, C. Volter, M.Sainz-Quevedo, P. Castorina, U. Ambrosetti, S. Berrettini, K. Frei, S.Tedin, J. Smith, M. Cruz Tapia, L. Cavalle, N. Gelvez, P. Primignani, E.Gomez-Rosas, M. Martin, M. A. Moreno-Pelayo, M. Tamayo, J.Moreno-Barral, F. Moreno, I. del Castillo, A multicenter study on theprevalence and spectrum of mutations in the otoferlin gene (OTOF) insubjects with nonsyndromic hearing impairment and auditory neuropathy,Hum Mutat 29(6) (2008) 823-31.

[6] A. Starr, Y. S. Sininger, H. Pratt, The varieties of auditoryneuropathy, J Basic Clin Physiol Pharmacol 11(3) (2000) 215-30.

[7] D. Beutner, A. Foerst, R. Lang-Roth, H. von Wedel, M. Walger, Riskfactors for auditory neuropathy/auditory synaptopathy, ORL JOtorhinolaryngol Relat Spec 69(4) (2007) 239-44.

[8] C. Madden, M. Rutter, L. Hilbert, J. H. Greinwald, Jr., D. I. Choo,Clinical and audiological features in auditory neuropathy, ArchOtolaryngol Head Neck Surg 128(9) (2002) 1026-30.

[9] H. Perez, J. Vilchez, T. Sevilla, L. Martinez, Audiologic evaluationin Charcot-Marie-Tooth disease, Scand Audiol Suppl 30 (1988) 211-3.

[10] D. Swanepoel, Auditory pathology in cri-du-chat (5p−) syndrome:phenotypic evidence for auditory neuropathy, Clin Genet 72(4) (2007)369-73.

[11] V. K. Manchaiah, F. Zhao, A. A. Danesh, R. Duprey, The geneticbasis of auditory neuropathy spectrum disorder (ANSD), Int J PediatrOtorhinolaryngol 75(2) (2011) 151-8.

[12] S. Delmaghani, F. J. del Castillo, V. Michel, M. Leibovici, A.Aghaie, U. Ron, L. Van Laer, N. Ben-Tal, G. Van Camp, D. Weil, F. Langa,M. Lathrop, P. Avan, C. Petit, Mutations in the gene encoding pejvakin,a newly identified protein of the afferent auditory pathway, causeDFNB59 auditory neuropathy, Nat Genet 38(7) (2006) 770-8.

[13] J. Suzuki, K. Hashimoto, R. Xiao, L. H. Vandenberghe, M. C.Liberman, Cochlear gene therapy with ancestral AAV in adult mice:complete transduction of inner hair cells without cochlear dysfunction,Sci Rep 7 (2017) 45524.

[14] E. Zinn, S. Pacouret, V. Khaychuk, H. T. Turunen, L. S. Carvalho,E. Andres-Mateos, S. Shah, R. Shelke, A. C. Maurer, E. Plovie, R. Xiao,L. H. Vandenberghe, In Silico Reconstruction of the Viral EvolutionaryLineage Yields a Potent Gene Therapy Vector, Cell Rep 12(6) (2015)1056-68.

[15] L. D. Landegger, B. Pan, C. Askew, S. J. Wassmer, S. D. Gluck, A.Galvin, R. Taylor, A. Forge, K. M. Stankovic, J. R. Holt, L. H.Vandenberghe, A synthetic AAV vector enables safe and efficient genetransfer to the mammalian inner ear, Nat Biotechnol 35(3) (2017)280-284.

[] Y. C. Lu, C. C. Wu, W. S. Shen, T. H. Yang, T. H. Yeh, P. J. Chen, I.S. Yu, S. W. Lin, J. M. Wong, Q. Chang, X. Lin, C. J. Hsu, Establishmentof a Knock-In Mouse Model with the SLC26A4 c.919-2A>G Mutation andCharacterization of Its Pathology, Plos One 6(7) (2011).

What is claimed is:
 1. A method for treating a method for treating anauditory neuropathy spectrum disorder in a subject comprisingtransferring a transgene via an adeno-associated virus (AAV) vector tothe subject; wherein the transgene is selected from the group consistingof Pjvk, PCDH15, GJB2, DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.
 2. Themethod of claim 1, wherein the transgene is Pjvk or PCDH15.
 3. Themethod of claim 1, wherein the AAV vector is an AAV vector comprising anAnc80 capsid protein.
 4. The method of claim 3, wherein the AAV vectoris a synthetic inner ear hair cell targeting adeno-associated virus(AAV) vector, wherein the vector encodes a capsid having at least about85% sequence identity to Anc80, and comprises a promoter selected fromthe group consisting of an Espin promoter, a PCDH15 promoter, a PTPRQpromoter and a TMHS (LHFPL5) promoter that directs expression ofharmonin-a, harmonin-b, or harmonin-c polypeptide.
 5. A construct fordelivering a transgene to a subject suffering from an auditoryneuropathy spectrum disorder, which comprises an adeno-associated virus(AAV) and the nucleic acids designated a transgene; wherein thetransgene is selected from the group consisting of Pjvk, PCDH15, GJB2,DIAPH3, PCDH9, SLC17A8, AIFM1, and OTOF.
 6. The construct of claim 5,wherein the transgene is Pjvk or PCDH15.
 7. The construct of claim 5,wherein the AAV vector is an AAV vector comprising an Anc80 capsidprotein.
 8. The construct of claim 7, wherein the AAV vector is asynthetic inner ear hair cell targeting adeno-associated virus (AAV)vector, wherein the vector encodes a capsid having at least about 85%sequence identity to Anc80, and comprises a promoter selected from thegroup consisting of an Espin promoter, a PCDH15 promoter, a PTPRQpromoter and a TMHS (LHFPL5) promoter that directs expression ofharmonin-a, harmonin-b, or harmonin-c polypeptide.